1. Field of the Invention
The present invention relates to a method for preparing mutant genes. More particularly, the present invention relates to a method for artificially and efficiently preparing unknown and known mutant genes in a short period of time.
2. Description of Related Art
Diverse and various evolution of organisims are brought about by mutation of genes coding structure and functions thereof.
Mechanisms causing mutations studied include an error of base complementarity upon replication of DNA, an error upon repair of damaged DNA, and transposable elements, but there still remain many unclear points. While the known structural changes include base substitution which causes a missense mutation or a nonsense mutation, a frameshift, and a change on chromosome such as deletion, duplication, inversion and translocation, the probability that these mutations spontaneously occur is very low.
For example, the probability of mispairing during one cycle of DNA replication is estimated at about 10.sup.-10 per base pair.
In order to prepare novel biological species or genes, therefore, it is necessary to artificially induce a DNA mutation.
The conventional practice to induce such a mutation has consisted of treating an individual or cells with a physical or chemical mutagen. Representative applicable physical mutagens include ultraviolet rays (UV) mainly causing base substitution, and ionizing radiations (X-rays, .gamma.-rays, etc.) inducing marked changes on the chromosome level such as deletion and translocation. The common features in these physical mutagens are simplicity, absence of the risk of residue, and possibility of quantitative handling, although the frequency of induced mutations is not very high. Known chemical mutagens include alkylating agents causing base substitution (eg., ethylmethane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and ethylnitrosourea), base analogs (eg., bromodeoxyuridine, and N4 aminocytidine) and intercalators causing a frameshift (such as ICR compounds). These chemical mutagens have a high frequency of induced mutations and are mainly used for inducing mutations of cultured cells. Particularly, alkylating agents such as EMS and MNNG are widely used because of the high frequency of induced mutations, but are defective in chemical stability, and tend to induce multiple mutations. It is therefore considered necessary to be careful in using them.
It is possible to increase the occurring frequency of mutations 100.about.1000 times by the use of these physical and chemical mutagens. It is however important to appropriately select a kind of mutagen according to the target mutation, and at the same time, to set an appropriate concentration of mutagen, treatment time, mutation expressing time and number of plates of propagated cells. The variables must further be set after repeated trials and errors for each cell covered.
For Escherichia coli and yeasts, on the other hand, methods for artificially preparing mutant strains by the use of gene manipulation are known, including, for example, a site-specific mutation using M13 phage for Escherichia coli, introduction of transposon using a suicide vector, and gene destruction using an insertional vector for yeasts. For Escherichia coli, there are also known a mutant strain (mut) which increases the mutation rate per generation, and a mutant strain (recA.sup.-, umuC.sup.-) which causes almost no mutation on the contrary.